Hydrogels are hydrophilic polymeric matrices that can support cell and tissue growth in three-dimension. With careful design, hydrogels serve not only as a 3D platform for studying cell behaviors, but also as scaffolds for culturing and differentiating stem/progenitor cells. We aim to develop a multifunctional hydrogel system that initially supports cell proliferation, but at a later stage can be "switched" into a microenvironment that promotes cell/tissue differentiation into a specific cell type. Our central hypothesis is that cell expansion and differentiation can be achieved in a single hydrogel matrix incorporated with dynamic biophysical and biochemical cues. We will test this hypothesis by developing a versatile hydrogel system using multiple cytocompatible thiol-ene "click" reactions. In the initial stage, we will design locally degradable thiol-ene gels to promote proliferation of PANC-1 cells (a pancreatic ductal epithelial cell line) (Aim 1). We will incorporate matrix metalloproteinase 2 (MMP-2) specific peptide substrates as hydrogel crosslinkers. This allows the gels to degrade locally (thus creating additional space for cell proliferation) by MMP-2 secreted from PANC-1 cells. The proliferation of encapsulated cells will be promoted by tuning cell-ECM and cell-cell interactions in hydrogels, as well as providing the encapsulated cells with diffusible soluble growth factors. Next, we will "switch" the cell-laden hydrogels from a "pro-proliferation" to a "pro-differentiation" microenvironment (Aim 2a). We will achieve this by performing a second thiol-ene click reaction to introduce pro-differentiation cues within the cell-laden hydrogels, thus promoting the formation of islet-like, insulin secreting cell clusters. The differentiation process will be a combinatorial result of using serum-free culture media, affinity-based recruitment of basic fibroblast growth factor (bFGF), and timely conjugation of glucagon-like peptide 1 (GLP-1). The differentiated cell clusters can be retrieved from the erodible gels (due to specific enzyme activity) for further characterization and biological/clinical applications (Aim 2b). The differentiated clusters are expected to form tight aggregates due to strong cell-cell interactions. Together, our strategies facilitate the formation of islet-like cell clusters with natural cell-cell interactions and normal insulin secretion profiles. PUBLIC HEALTH RELEVANCE: This proposal aims to design biomaterial devices that incorporate signals to promote the proliferation of epithelial cells and to enhance the differentiation of these cells into insulin-producing cells. If successful, this strategy will provide alternative cell sources to benefit type 1 diabetic patients.